Investigation Of Anti-inflammatory Effect Of Chlorogenic Acid And Its Molecular Mechanism Based On MiR-155/NLRP3 Inflammasome Signaling Pathway

Objective The activation of inflammasome is an important signaling pathway in inflammatory response.The activation of inflammasome occured in a variety of inflammatory diseases,such as colitis.We investigated whether miR-155 regulates inflammasomes and chlorogenic acid exerts anti-inflammatory effects in vivo and in vitro.Methods(1)The inflammatory model of RAW264.7 macrophage was established by ATP stimulation after LPS prestimulation.The methods of MTT and the release of lactate dehydrogenase were used to observe the cell growth,the release of IL-1β was detected by ELISA,immunofluorescence was used to detect the expression of inflammatory signaling pathway related proteins in the inflammatory cells.(2)qRT-PCR was used to validate the target interaction between miR-155,NLRP3 and IL-1β in the inflammatory model of RAW264.7 macrophage.(3)Chlorogenic acid regulated the inflammasome.The inflammatory model of RAW264.7 macrophage was established after adding chlorogenic acid.MTT method and detecting the release of lactate dehydrogenase were used to observe the cell growth effects,ELISA was detected the release of IL-1β,Western blot and immunofluorescence were used to detect the inflammatory actived signal molecules of p-NF-κB,NLRP3,ASC,Caspase-1 p45,Caspase-1 p20 and the cytokine of pro-IL-1β,cleaved-IL-1β,pro-IL-18,cleaved-IL-18.qRT-PCR was detected the expression of miR-155.To compare chlorogenic acid with miR-155 inhibitor on the effects of miR-155,IL-1β and NLRP3 mRNA in the inflammatory cells model,Western blot was used to detect transfected miR-155 inhibitor cells the inflammatory actived signal molecules of p-NF-κB,qRT-PCR was tested the effects of miR-155 mimics and chlorogenic acid on miR-155 and IL-1β mRNA in RAW264.7 cells.The effect of miR-155 inhibitor and mimics on the secretion of IL-1β in RAW264.7 macrophages was detected by ELISA.(4)Chlorogenic acid improved colitis by regulating inflammasome.The colon length of dextran sulfate sodium(DSS)induced murine colitis was compared.The colonic morphology of mice was observed by HE,and the positive expression of macrophage in the colon of mice was observed by immunohistochemistry.Western blot was used to detect the inflammatory actived signal molecules of p-NF-κB,NLRP3,ASC,Caspase-1 p45,Caspase-1 p20 and the cytokine of pro-IL-1β,cleaved-IL-1β,pro-IL-18,cleaved-IL-18.The effect of chlorogenic acid on miR-155 in colon tissue of DSS model mice was detected by qRT-PCR.Result(1)LPS induced the activation of TLR4 signaling pathway.After adding ATP,the activation of inflammasome in RAW264.7 macrophages was significantly increased.The release of lactate dehydrogenase and the release of IL-1β were increased.The expression of IL-1β and NLRP3 were increased.(2)The expression of miR-155 was increased in the inflammatory model of RAW264.7 macrophage.With transfection miR-155 inhibitor,the expression of miR-155 and NLRP3 mRNA were decreased.(3)Chlorogenic acid could decrease the release of lactate dehydrogenase and the release of IL-1β in the inflammatory model of RAW264.7 macrophage.The inflammatory actived signal molecules of p-NF-κB,NLRP3,ASC,Caspase-1 p45,Caspase-1 p20 and the cytokine of pro-IL-1β,cleaved-IL-1β,pro-IL-18,cleaved-IL-18 were decreased.Chlorogenic acid could reduce the expression of miR-155,NLRP3,IL-1β mRNA and p-NF-κB protein in the inflammatory model of RAW264.7 macrophage.With transfection miR-155 mimics,the expression of miR-155,IL-1β mRNA and p-NF-κB protein was reduced.(4)The colon length of dextran sulfate sodium(DSS)induced murine colitis were reduced in vitro,the number of macrophages in colon of murine colitis were increased and the expression of p-NF-κB,NLRP3,ASC,Caspase-1 p45,Caspase-1 p20,pro-IL-1β,cleaved-IL-1β,pro-IL-18,cleaved-IL-18 protein was higher than the normal group.Chlorogenic acid could improve colitis in mice and could decrease the expression of inflammasome related proteins and miR-155.Conclusion miR-155 promotes the activation of inflammasome through NF-κB and IL-1β.Chlorogenic acid can inhibit the production of inflammasome and inflammatory factors in vivo and in vitro,and has good anti-inflammatory effect.Its anti-inflammatory mechanism may be related to the regulation of miR-155 expression.